Impact of a single nucleotide polymorphism on the 3D protein structure and ubiquitination activity of E3 ubiquitin ligase arkadia

Single nucleotide polymorphisms (SNPs) are genetic variations which can play a vital role in the study of human health. SNP studies are often used to identify point mutations that are associated with diseases. Arkadia (RNF111) is an E3 ubiquitin ligase that enhances transforming growth factor-beta (TGF-β) signaling by targeting negative regulators for degradation. Dysregulation of the TGF-β pathway is implicated in cancer because it exhibits tumor suppressive activity in normal cells while in tumor cells it promotes invasiveness and metastasis. Τhe SNP CGT > TGT generated an amino-acid (aa) substitution of Arginine 957 to Cysteine on the enzymatic RING domain of Arkadia. This was more prevalent in a tumor than in a normal tissue sample of a patient with colorectal cancer. This prompted us to investigate the effect of this mutation in the structure and activity of Arkadia RING. We used nuclear magnetic resonance (NMR) to analyze at an atomic-level the structural and dynamic properties of the R957C Arkadia RING domain, while ubiquitination and luciferase assays provided information about its enzymatic functionality. Our study showed that the R957C mutation changed the electrostatic properties of the RING domain however, without significant effects on the structure of its core region. However, the functional studies revealed that the R957C Arkadia exhibits significantly increased enzymatic activity supporting literature data that Arkadia within tumor cells promotes aggressive and metastatic behavior

A helical region in the C terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins

An NMR-derived model for the solution structure of oxidized Thermotoga maritima 1[Fe4-S4] ferredoxin.

The solution structure of the 60-residue 1[Fe4-S4] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron-sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1[Fe4-S4] ferredoxins from Desulfovibrio gigas and Desulfovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the T. maritima protein. In particular, the T. maritima protein displayed a beta-sheet structure made up of strands located at the very NH(2) and COOH termini of the protein, and an internal alpha-helix. The internal beta-sheet observed in the D. gigas and D. africanus ferredoxins could not be confirmed in T. maritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure

A refined model for [Fe3S4](0) clusters in proteins

The Bacillus schlegelii ferredoxin was characterized by electrochemical measurement